Cameron, S., Chen, Y. and Rao, Y. (2016). Borderless regulates glial extension and axon ensheathment. Dev Biol [Epub ahead of print]. PubMed ID: 27131624Summary:
Ensheathment of axons by glial processes is essential for normal brain function. While considerable progress has been made to define molecular and cellular mechanisms underlying the maintenance of axon ensheathment, less is known about molecular details of early events for the wrapping of axons by glial processes in the developing nervous system. This study investigated the role of the transmembrane protein Borderless (Bdl) in the developing Drosophila visual system. Bdl belongs to the immunoglobulin (Ig) superfamily, and its in vivo function is unknown. This study shows that Bdl is expressed in wrapping glia (WG) in the developing eye disc. Cell-type-specific transgene rescue and knockdown indicate that Bdl is specifically required in WG for the extension of glial processes along photoreceptor axons in the optic lobe, and axon ensheathment. These results identify Bdl as a novel glia-specific cell-surface recognition molecule in regulating glial extension and axon ensheathment.

Chen, Y., Cameron, S., Chang, W. T. and Rao, Y. (2017). Turtle interacts with borderless in regulating glial extension and axon ensheathment. Mol Brain 10(1): 17. PubMed ID: 28535795Summary:
Proper recognition between axons and glial processes is required for the establishment of axon ensheathment in the developing nervous system. Recent studies have begun to reveal molecular events underlying developmental control of axon-glia recognition. Previous work has shown that the transmembrane protein Borderless (Bdl) is specifically expressed in wrapping glia (WG), and is required for the extension of glial processes and the ensheathment of photoreceptor axons in the developing Drosophila visual system. The exact mechanism by which Bdl mediates axon-glia recognition, however, remains unknown. This study presents evidence showing that Bdl interacts with the Ig transmembrane protein Turtle (Tutl). Tutl is specifically expressed in photoreceptor axons. Loss of tutl in photoreceptors, like loss of bdl in WG, disrupts glial extension and axon ensheatment. Epistasis analysis shows that Tutl interacts genetically with Bdl. Tutl interacts with Bdl in trans in cultured cells. It is proposed that Tutl interacts with Bdl in mediating axon-glia recognition for WG extension and axon ensheathment.

The presence of numerous axons and dendrites in the neuropils of the developing CNS makes it a daunting task for establishing specific synaptic connections. Studies over the last two decades have identified a number of cell-surface recognition molecules that mediate specific neurite-neurite interactions for circuit assembly. That many cell-surface recognition molecules are present broadly in developing neuropils throughout embryonic development, however, raises the question how the action of cell-surface recognition molecules is modulated temporally to ensure accuracy in circuit formation (Cameron, 2013).

The assembly of visual circuits in Drosophila is an attractive model for understanding the general mechanisms underlying spatiotemporal control of neurite-neurite interactions (Hadjieconomou, 2011; Melnattur, 2011). The Drosophila adult visual system is comprised of the compound eye and the optic lobe. The compound eye consists of ~800 ommatidia, each containing six outer photoreceptor neurons (R1-R6) for processing motion and two inner photoreceptor neurons (R7 and R8) for processing color. R1-R6 axons form synaptic connections in the superficial lamina layer, and R7 and R8 axons project through the lamina into the deeper medulla layer, where they are organized into ~800 regularly spaced columns. Each R7 and R8 axon from the same ommatidium terminate in a topographic manner in two synaptic layers within the same column. The R8 axon terminates within the M3 layer, and the R7 axon terminates in the deeper M6 layer (Cameron, 2013).

The role of a novel Ig-superfamily transmembrane protein Borderless (Bdl) in Drosophila was investigated in this study. Bdl is expressed in the developing visual system, and functions as a cell-surface recognition molecule to mediate neurite-neurite interactions. The receptor tyrosine phosphatase LAR and the Ig-superfamily transmembrane protein Tutl are key regulators of Bdl-mediated axon-axon interactions in controlling synaptic-layer selection and axonal tiling, respectively. The results shed new light on spatiotemporal control of cell-surface recognition molecules for coordinating circuit assembly (Cameron, 2013).

Tiling and self-avoidance, two cellular mechanisms discovered in the early 1980s, are important for patterning neuronal circuitry. Previous studies have identified several cell-surface recognition molecules, such as Dscam, Tutl, Protocadherins, MEGF10, and MEGF11, that mediate homotypic neurite-neurite interactions in tiling and self-avoidance. These cell-surface recognition molecules may act by mediating homotypic repulsion or de-adhesion between adjacent same-type neurites. For instance, molecular and genetic analyses of fly Dscam1 support a role for Dscam1 in mediating homotypic repulsion in dendritic self-avoidance, whereas mammalian Dscams appear to mediate de-adhesion by interfering with some unknown cell-type-specific cell adhesion molecules (Fuerst, 2009). The exact mechanisms by which these cell-surface recognition molecules mediate homotypic repulsion or de-adhesion, however, remains elusive (Cameron, 2013).

Several lines of evidence implicate Bdl as a target of Tutl in regulating R7 axonal tiling. First, overexpression of Bdl induced an R7 tiling phenotype similar to that in tutl mutants. Second, Tutl associates with Bdl in cultured cells. And third, loss of bdl rescued the tiling phenotype in tutl mutants. It is proposed that Tutl-mediated surface recognition counteracts the affinity between adjacent R7 axonal terminals by interacting with Bdl. The association of Tutl with Bdl may downregulate the level and/or adhesive activity of Bdl, thus allowing the separation of adjacent R7 axonal terminals. Since co-overexpression of Tutl and Bdl did not affect Bdl-mediated cell-cell aggregation in culture nor the Bdl-overexpression-induced tiling phenotype in flies, it is speculated that the regulation of Bdl by Tutl requires the involvement of additional regulatory molecules. Future studies are needed to determine the exact mechanism by which Tutl downregulates the function of Bdl. It will also be of interest to determine whether other cell-surface recognition molecules implicated in tiling and self-avoidance (e.g., Dscam and Protocadherins), function similarly to modulate certain cell adhesion molecules (Cameron, 2013).

The receptor tyrosine phosphatase LAR and its mammalian homologs have been shown to play important roles in axon guidance, neuronal target selection, and presynaptic development. In the developing Drosophila visual system, LAR is required for target selection of R1-R6 axons in the lamina, and synaptic-layer selection of R7 axons in the medulla. The action of LAR in R7 synaptic-layer selection reportedly involves both stabilization of axon-target interactions and down-regulation of adhesion between R7 and R8 axons. LAR-mediated axon-target interactions may involve the binding between LAR on R7 axons and an unknown ligand in the target layer, which in turn modulates the interaction between LAR and its cytoplasmic domain-binding partner Liprin to stabilize axon-target interactions. It is also reported that LAR negatively regulates an unknown cell adhesion molecule to decrease adhesion between R7 and R8 axons for facilitating synaptic-layer selection of R7 axons (Cameron, 2013).

The current results suggest strongly that LAR downregulates adhesion between R7 and R8 axons by negatively regulating Bdl. That LAR inhibited Bdl-mediated cell-cell adhesion without affecting the level of Bdl suggests that LAR inhibits adhesive activity of Bdl. Although the role of LAR in mediating axon-target interactions requires its binding to Liprin via the cytoplasmic domain, negative regulation of Bdl by LAR appears to involve a Liprin-independent mechanism. This is supported by in vitro analysis showing that a LAR mutant lacking the cytoplasmic domain also inhibited Bdl-mediated adhesion. Consistently, a previous study showed that R8-specific expression of a truncated LAR mutant lacking the cytoplasmic domain in LAR mutants could partially rescue the R7 mistargeting phenotype. LAR may directly modulate Bdl to downregulate R7-R8 adhesion, or act indirectly by interacting with other proteins. Future studies are needed to distinguish between these possibilities (Cameron, 2013).

Although negative regulation of Bdl-mediated axon-axon interactions is necessary for R7 synaptic-layer selection and tiling, it remains unclear how the presence of Bdl contributes to the formation of the R-cell axonal projection pattern in the fly visual system. Cell adhesion molecules, such as NCAM/FasII and L1-CAM/Neuroglian, have been shown to mediate selective fasciculation in axonal pathfinding. Similarly, Bdl-mediated axon-axon interactions may facilitate the projections of R7 and/or R1-R6 axons along the pioneer R8 axon. That the R-cell projection pattern remained normal in bdl mutants may be due to the presence of redundant genes. Functional redundancy among different cell adhesion molecules seems to be common in the developing nervous system, which may account for no or subtle phenotypes in mutants defective in a number of cell adhesion molecules (Cameron, 2013).

In conclusion, this study study identifies Bdl as a novel and important regulator of neurite-neurite interactions in the developing visual system. Tuning of Bdl-mediated axon-axon interactions in axonal tiling and synaptic-layer selection presents an excellent example for modulating the action of cell adhesion molecules in ensuring accuracy in circuit assembly. It is highly likely that similar mechanisms are employed for circuit assembly in mammalian nervous systems (Cameron, 2013).